This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Coxsackievirus B3 (CVB3) is the leading cause of viral myocarditis, causes pancreatitis and plays a role in type I diabetes. Like all picornaviruses, CVB3 translates its RNA genome using an internal ribosome entry site (IRES), whereby ribosomes are recruited directly to an initiation codon by recognizing a highly structured RNA element in the 5'nontranslated region (5'NTR). The overall goal of this research is to understand the structure and function of the 5'NTR and its associated IRES. The specific objective of this proposal is to explore the RNA elements and the RNA-protein interactions that determine virulence in CVB3. The two specific aims of the previous grant were accomplished, confirming the hypothesis that the IRES folds into a stable structure that is required for proper function. As proposed in specific aim 1, the structure of a wild type CVB3 IRES was determined. As proposed in specific aim 2, differences that mediate the virulence phenotype have been mapped. The goal of the current research is to define the minimal RNA elements that determine virulence and explore the RNA-protein interactions that underlie IRES function. Two specific aims will test the hypothesis that small, independently folding RNA structures in the 5'NTR provide recognition features for cellular and viral proteins that together determine viral virulence. In specific aim 1, the solution structure of RNA domains previously identified to be virulence determinants will be determined in virulent and non-virulent variants of the genome. These variants will include naturally occurring sequences, site-directed mutants and chimeric constructs. Our established methods of base-specific chemical modifying agents will probe the accessibility of nucleotides in the folded RNA domains. In specific aim 2 the molecular mechanism of IRES function will be explored by studies of RNA-protein interactions. Probing studies of RNA-protein complexes using our established chemical modification techniques will provide insight into the conformational dynamics and functional states of the 5'NTR. These results will aid in the search for effective antivirals and vaccines, not only for CVB3 but also for a host of other disease-causing picornaviruses.